Evolutionary dynamics of canine kobuvirus in Vietnam and Thailand reveal the evidence of viral ability to evade host immunity

Canine kobuvirus (CaKoV) is a pathogen associated with canine gastrointestinal disease (GID). This study examined 327 rectal swabs (RS), including 113 from Vietnam (46 healthy, 67 with GID) and 214 from Thailand (107 healthy and 107 with GID). CaKoV was detected in both countries, with prevalences of 28.3% (33/113) in Vietnam and 7.9% (17/214) in Thailand. Additionally, CaKoV was found in both dogs with diarrhea and healthy dogs. CaKoV was mainly found in puppies under six months of age (30.8%). Co-detection with other canine viruses were also observed. The complete coding sequence (CDS) of nine Vietnamese and four Thai CaKoV strains were characterized. Phylogenetic analysis revealed a close genetic relationship between Vietnamese and Thai CaKoV strains, which were related to the Chinese strains. CDS analysis indicated a distinct lineage for two Vietnamese CaKoV strains. Selective pressure analysis on the viral capsid (VP1) region showed negative selection, with potential positive selection sites on B-cell epitopes. This study, the first of its kind in Vietnam, provides insights into CaKoV prevalence in dogs of different ages and healthy statuses, updates CaKoV occurrence in Thailand, and sheds light on its molecular characteristics and immune evasion strategies.


Genetic characterization of the CaKoV in Vietnam and Thailand
The CDS of CaKoV from Vietnam (9 sequences) and Thailand (4 sequences) were successfully characterized and deposited in GenBank database under accession numbers PP320358-PP320370.The CaKoV genome spanned a length of 7571 nt, featuring a single ORF that encoded 2444 aa.The ORF initiated with the methionine codon (Met) and ended with the alanine codon (Ala).Details outlining the structure of the CaKoV genome, as identified in this study, are presented in Table 2. Additionally, information about the RS samples from dogs that were used to assemble the complete CaKoV genome is provided (Supplementary Table 3).The nucleotide and amino acid similarities between the CaKoV sequences from Vietnam and those from Thailand in this study were compared with strains previously available in the GenBank database, originating from diverse countries such as Thailand, China, Germany, India, South Korea, the UK, Australia, Tanzania, Brazil, and the United States (Fig. 1, Supplementary Table 4, Supplementary Table 9).Within the CDS of Vietnamese CaKoV sequences, a similarity of 95-97.3% in nt and 92-96.4% in aa was observed.The four CDS of Thai CaKoV sequences exhibited a similarity of 96.3-99.9% in nt and 93.6-99.9% in aa.When comparing CDS of CaKoV obtained from Vietnam and Thailand, they displayed similarities of 94.6%-96.5% in nt and 91.9%-95% in aa.Remarkably, they showed the highest nt similarity with the Thai strains from the previous study (MK201176-79) and the Chinese strain (MN449341), while exhibiting the lowest nt similarity to Tanzanian strains (KM068048-51) (Fig. 1, Supplementary Table 4).As expected, the VP1 gene was the most variable region for CaKoV, with nt similarities ranging from 81.6 to 96.5% and aa similarities ranging from 81.1 to 98.5% (Supplementary Table 4).Notably, no recombination events were identified in the CaKoV strains obtained in this study.

Phylogeny of the Vietnamese and Thai CaKoV
The phylogenetic analysis, based on the nucleotide sequences of the CDS, P1-P2-P3 region, VP, and 3D genes of nine CaKoV Vietnamese strains and four CaKoV Thai strains, is presented in Fig. 2. The tree based on the CDS indicated a close genetic relationship between Vietnam and Thailand strains, showing genetic affinities with Chinese strains.Interestingly, two Vietnamese strains (PP320366_V162/Vietnam/2022 and PP320370_V196/ Vietnam/2022) formed a distinct subgroup, sharing genetic characteristics with both Chinese and Thai strains.The phylogenetic tree based on the P1 region exhibited similar characteristics to the CDS tree; however, the PP320365_V156/Vietnam/2022 strain formed a separate clade.Furthermore, the phylogenetic tree based on the VP1 gene, known for its high variability and belonging to the P1 region, suggested that the two CaKoV strains from Vietnam (PP320366_V162/Vietnam/2022 and PP320370_V196/Vietnam/2022) were closely related to strains MN337880 (isolated from foxes in Germany), and KM068049-068051 (isolated from wild carnivores in Tanzania), rather than strains isolated from domestic dogs in Asia and the Americas.Examining the P2 and P3 regions, the phylogenetic tree revealed that the Vietnamese and Thai strains in this study tended to cluster together, sharing genetic characteristics with strains from China, India, Australia, and previous Thai strains.They were separated from strains in Europe, the USA, Brazil, and Tanzania.For the 3D gene of CaKoV, belonging www.nature.com/scientificreports/ to the P2 region, the phylogenetic tree displayed less genetic variation, with the strains in this study forming a cohesive subgroup.

B-cell epitope prediction for Vietnamese and Thai CaKoV
The VP1 capsid gene of CaKoV was employed for the prediction of both continuous and discontinuous B-cell epitopes.Multiple methods were utilized for predicting continuous B-cell epitopes, and the outcomes from each method were visualized through distinct charts and tables (refer to Table 3 and Fig. 3).Residues with scores above the threshold (depicted by the red line on the chart) were considered indicative of having a higher likelihood of being potential epitopes.Additionally, the overlapping aa regions from all plots, based on the results of all prediction methods, served as the basis for suggesting potential continuous B-cell epitopes (Fig. 3).
The ElliPro tool identified six discontinuous epitopes from the CaKoV VP1 gene capsid structure (Fig. 4).These epitopes varied in size from 8 to 36 residues, with scores ranging from 0.608 to 0.848.Information on the residues associated with discontinuous epitopes, including the quantity, positions, and scores, were detailed in Supplementary Table 8.Notably, the ElliPro predictions for discontinuous B-cell epitopes, based on the CaKoV VP1 region, overlapped with the linear B-cell epitopes.Furthermore, the Ellipro chart depicting continuous B-cell epitopes (Fig. 4g) revealed yellow areas, aligning with antigenic characteristics as indicated by the linear epitope predictions from the BepiPred linear epitope 2.0 method (Fig. 3b).

Selection pressure for Vietnamese and Thai CaKoV
The analysis of the dN and dS ratios derived from the CaKoV VP1 gene alignment was conducted using the MEME and SLAC methods.Both analyses suggested that the VP1 gene of CaKoV from Vietnam and Thailand underwent negative selection pressure (with dN/dS < 1), indicating a tendency towards purifying or stabilizing   www.nature.com/scientificreports/selection.However, it is noteworthy that potential positive selection sites were also identified, implying regions where positive selection might be occurring (Table 4).
The analysis of selective pressure on the VP1 region of CaKoV revealed specific codon positions that were under positive selection.For Vietnamese CaKoV, the 204th and 206th codon positions were identified as positive selected, with the 206th codon being detected by both MEME and FUBAR methods, while the 204th codon was identified only by the FUBAR method.For Thai CaKoV, four codon positions (69th, 82nd, 138th, and 206th) were found to be positively selected, but they were only detected by the MEME method.The analysis also explored the relationship between the impact of selective pressure and B-cell epitopes.In both Vietnamese and Thai CaKoV, there was a positively selected position at the 206th codon, which corresponds to positions within predicted linear and conformational B-cell epitopes; the Thai CaKoV strain exhibited the additional presence of two positively selected positions, 69th and 138th codons.This suggests a potential interplay between selective pressure and the regions of the virus that are recognized by the immune system.

Discussion
This study revealed a prevalence of CaKoV in Vietnam and provided an update status of CaKoV in Thailand, showing a lower prevalence compared to a previous study 28 .Our investigation found that the highest detection rate of CaKoV was in dogs under 6 months old, aligning with similar patterns observed in previous reports, where the highest prevalence occurred in dogs under 4 months old in Japan (Soma et al. 27 ) and 1 year old in Thailand 28 .Notably, human studies have also reported a higher incidence of Human Aichi Virus (AiV) infection in children [35][36][37] .In addition to affecting young dogs, CaKoV was found across various age groups in this study, consistent with findings in both humans 38 and dogs 28 .However, a study in Japan reported that CaKoV was not detected in dogs over 5 months old 27 .These variations suggest that the prevalence of CaKoV may differ between countries, influenced by factors such as sample sizes and the specific age groups sampled.
The detection of CaKoV in both diarrhea and non-diarrhea dogs aligns with similar findings in studies from China 2,23 , Korea 26 , Japan 27 .The CaKoV prevalence in dogs with diarrhea tends to be higher than in dogs www.nature.com/scientificreports/without diarrhea, but due to the small sample size in this study, further research is needed to clarify this point.Moreover, in dogs with diarrhea, CaKoV was identified either as a single detection or in co-detection with other common enteric viruses.This pattern is consistent with previous research where CaKoV was found either as a sole detection 2,19,22,26,27 or in co-detection with viruses such as CPV2, rotavirus, CCoV, CDV, CaAstV, and Canine bocavirus 2,19,20,22,[25][26][27]39 , as well as bacteria or parasites like Campylobacter upsaliensis, Toxocara canis, Toxocara leonina 20 and giardiasis 32 . Siilarly, studies on AiV have shown a high rate of single AiV infection, suggesting www.nature.com/scientificreports/its potential role in gastrointestinal tract diseases in humans 38 .However, the true pathogenic role of CaKoV remains uncertain.It is unclear whether CaKoV can independently cause disease or if it collaborates with other enteric pathogens to induce gastroenteritis in dogs 40 . A gloal study suggests that CaKoV might play a sporadic pathogenic role in specific dog populations or interact with other enteric pathogens 29 .Therefore, additional observational studies or experimental infections are crucial to understanding the pathogenic mechanism of CaKoV in the gastrointestinal tract and its interactions with other pathogens 4 .However, the inability to cultivate CaKoV poses a significant challenge in the pathobiological research of this virus 20,26 .
In the comparison of CaKoV structure between this study and AiV, it was noted that the proline-rich region at aa position 50-63 of the VP0 gene of CaKoV was absent.Only the proline-rich region at aa position 228-240 (P 228 XPPPPXPPXPXP 240 ), which belong to the VP1 gene, showed similarity between CaKoV and AiV 41 .This distinctive proline-rich region plays a crucial role in mediating host-virus interactions, precise localization of adhesion, infectivity, and viral pathogenesis 42,43 .In the phylogenetic analysis, the results of this study revealed a significant tendency for strains from the same country to cluster together, consistent with previous reports 19,29,34,39 .The phylogenetic tree of CDS indicated that the sequences from China, Vietnam and Thailand formed an independent group, separated from sequences found in Africa, the United States, and Europe 28,39 .However, two Vietnamese CaKoV strains (PP320366_V162/Vietnam/2022, PP320370_V196/Vietnam/2022) showed a tendency to form a new monophyletic subgroup.Moreover, the VP1 gene of strains PP320366_V162/ Vietnam/2022 and PP320370_V196/Vietnam/2022 exhibited genetic heterogeneity compared to other Vietnamese CaKoV strains.Importantly, recombination events were not recorded for the Vietnamese and Thai CaKoV strains in this study.These findings suggest that strains PP320366_V162/Vietnam/2022 and PP320370_V196/ Vietnam/2022 may have regional characteristics or may be ancestral strains.However, the availability of complete CaKoV genome sequences was relatively limited, and increasing the number of CaKoV CDSs in various countries would help clarify this issue.
B-cell epitopes, or antigenic determinants, are distinct clusters of amino acids on an antigen that are recognized by secreted antibodies or B-cell receptors, inducing cellular or humoral immune responses 44,45 .The analyis and prediction of B-cell epitopes were conducted using IEDB resources, focusing on the VP1 gene region of kobuvirus.This region is essential for receptor interaction, exhibits high immunogenicity among picornavirus capsid proteins, and experiences significant selective pressure 7,15,[46][47][48] .The impact of selective pressure on the host immune response to CaKoV was explored.The analysis revealed that negative selection predominantly influenced the evolutionary dynamics of the VP1 gene region of CaKoV in Vietnam and Thailand.This suggests that external selection pressure may not be closely linked to the presence of mutations.Negative selection reflects an inclination to maintain the overall antigenic stability of CaKoV, indicating a low evolutionary rate and a delicate balance between virus-host interaction and survival ability [49][50][51][52][53] .However, potential positive selection positions in the VP1 gene were still identified.This suggests that selective pressure tends to alter the B-cell epitope of CaKoV, making it less conserved.Consequently, the host's immune response to CaKoV may undergo changes, potentially leading to immune evasion.
In summary, this study found that CaKoV was prevalent in both diarrheic and non-dirrheric dogs of all ages, with the highest prevalence observed in dogs under 6 months old.CaKoV was not only detected as a single infection but also in co-detection with other common gastrointestinal viral pathogens.The phylogenetic tree and genomic characteristics analyses revealed a close relationship between Vietnamese and Thai CaKoV strains, and they were also closely related to Chinese strains.Notably, Vietnamese strains formed a unique sub-branch.Selective pressure analysis showed that negative selection was the predominant force shaping the evolution of Vietnamese and Thai CaKoVs.However, potential positive selection sites were identified within the VP1 gene, particularly in regions associated with predicted B-cell epitopes.This suggests that while there is overall pressure to maintain stability, certain regions of the virus might be evolving under positive selection, possibly impacting host immune responses.The need for further research to elucidate the pathogenic mechanisms and immune responses associated with CaKoV infections in dogs.

Sample collection and RNA extraction
A total of 327 RS samples were collected from pet dogs visiting veterinary clinics in Vietnam and Thailand between August 2021 and August 2022.This comprised 113 samples from Ho Chi Minh City, Vietnam (46 from healthy dogs and 67 from GID dogs) and 214 samples from Bangkok and Nakhon Si Thammarat Province,  54 .
The animal sampling and research protocols for this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) (No. 2231006) and the Institutional Biosafety Committee (IBC) (No. 2131019) of Chulalongkorn University (Bangkok, Thailand).Approval was also obtained from the Animal Ethics Committee (AEC) (No.NLU-220217) of Nong Lam University (Ho Chi Minh City, Vietnam).All procedures were performed in accordance with the relevant guidelines and regulations.The authors complied with the ARRIVE guidelines.Written informed consent was obtained from the owners for the participation of their animals in this study.
The RS sampling procedure involved inserting a sterile disposable cotton swab (Puritan, Guilford, USA) into the dog's rectum, followed by immersion in a 1.5 mL Eppendorf tubes containing 0.7 mL of 1% (v/v) sterile phosphate-buffered salt (PBS) at pH 7.4.The samples were stored at − 80 °C until the extraction step.Viral RNA extraction was carried out using the Nucleic Acid Extraction Kit II (Geneaid, Ltd., Taipei, Taiwan) according to the manufacturer's protocol.Subsequently, the extracted RNA samples were quantified and assessed for quality using a Nanodrop® Lite spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at the absorbance ratio A260/A280 and stored at − 80 °C until further use.

CaKoV detection
The extracted RNA samples underwent screening for the presence of CaKoV using the reverse transcription-polymerase chain reaction (RT-PCR) method with specific primers.These primers were self-designed to target the 3D gene region of CaKoV, referencing the available CaKoV sequence JQ911763 (Supplementary Table S1).The total volume was adjusted to 25 μl for the QIAGEN® One-Step RT-PCR Kit (Qiagen GmbH, Hilden, Germany), including 5 μl of QIAGEN 5 × buffer, 1 μl of 10 mM dNTP Mix, 2 μl of 0.6 μM forward and reverse primers, 1 µl of enzyme mixture, 3 µl of extracted RNA, and 11 µl of distilled water.
The thermal cycling conditions were conducted on a PCR thermocycler (SensoQuest GmbH, Göttingen, Germany), involving a complementary DNA (cDNA) synthesis step at 50 °C for 30 min, followed by inactivation of reverse transcription (RT) enzyme and initial PCR activation step at 95 °C for 15 min.Subsequently, 40 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, and extension at 72 °C for 1 min were carried out, followed by a final extension step at 72 °C for 10 min.The positive control for CaKoV was synthesized using GeneArt™ Strings™ DNA fragments based on the 3D region of strain JQ911763 (Thermo Fisher Scientific GmbH, Darmstadt, Germany).A no-template control (NTC) was employed as a negative control.PCR products were visualized using the QIAxcel® DNA Screening Kit (Qiagen GmbH, Hilden, Germany) and the Qiaxcel® highresolution capillary electrophoresis device (Qiagen GmbH, Hilden, Germany).Installation and analysis procedures were followed a previous report protocol 55 .The presence of the 438 bp amplicon product was considered as CaKoV positive.Subsequently, the PCR products were sent for genetic sequencing using a NGS-based method (Celemics, Inc., Seoul, Korea) to double-confirm the presence of CaKoV.The derived nucleotide sequences were analyzed and aligned with previously described CaKoV sequences depositing in the GenBank database using BLASTn analysis.CaKoV-positive samples were also tested for co-detection with other common canine enteric viruses by PCRs, including CPV 56 , CDV 57 , CCoV 58 , and CaAstV 59 .

CaKoV whole genome characterization
CaKoV-positive samples, initially identified by CaKoV-PCR screening, underwent further investigation for full-length genome analysis using multiple PCR assays.Seven primer pairs, self-designed based on nucleotide alignments from previously described CaKoV sequences available in the GenBank database (Supplementary Table 1), were employed to amplify the full-length CaKoV genome.The initial step involved the construction of cDNA from the extracted RNA samples, using the Omniscript® Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany).For cDNA synthesis, the final volume was 20 μl, comprising 2 μl of 10 × RT buffer, 2 μl of 5 mM dNTP mixture, 1 μl of 10 μM random primer (Promega, Madison, Wisconsin, USA), 1 μl of RNase-free water, 0.75 μl of 1 × buffer, 0.25 μl of RNase inhibitor (10 units/μl), 1 μl of Omniscript reverse transcriptase, and 12 μl of extracted RNA sample.
Subsequently, the PCR reaction was performed with a final volume of 25 μl, including 3 μl of cDNA, 1 μl of 1 μM for each forward and reverse primers, 12.5 μl of GoTaq® Green Master Mix (Promega, Madison, Wisconsin, USA), and 7.5 μl of distilled water.The thermal cycling conditions included an initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 1 min, 55-58 °C for 1 min, and 72 °C for 1 min, and then a final extension at 72 °C for 10 min.PCR products were visualized using the QIAxcel® DNA Screening Kit, as mentioned above.Positive amplicons were then subjected to genomic sequencing according to the protocol described above.Subsequently, the obtained gene sequences were aligned and assembled using the BioEdit software package version 7.2 with the ClustalW function.

Phylogenetic and genetic analyses
Genetic analysis involved comparing the similarity of the CaKoV nucleotide sequence obtained from this study with the CaKoV sequences available in the database.Phylogenetic trees were constructed based on the complete coding sequence (CDS), P1-P2-P3 regions, and two genes, VP1 and 3D of CaKoV.The MEGA software package version 10.0 was used for this purpose.The software utilized the Find Best DNA/Protein Models (ML) option to

Figure 1 .
Figure 1.Heatmap of the nucleotide and amino acid similarity of complete coding sequences of canine kobuvirus (CaKoV) among isolates from Vietnam and Thailand, along with reference strains.(a) The similarity of nucleotide sequences; (b) the similarity of amino acid sequences.

Figure 2 .
Figure 2. Phylogenetic trees of canine kobuvirus (CaKoV) for various regions: (a) the complete coding sequences (CDSs), (b) P1 region, (c) P2 region, (d) P3 region, (e) VP1 gene, and (f) 3D gene.Trees of CDSs, P1, P2, and P3 region were constructed using MEGA v10.0 with the neighbor-joining algorithm employing the general time-reversible model.For VP1 and 3D genes, the trees were constructed with the Hasegawa-Kishono-Yano method, incorporating a gamma distribution and invariable sites (G + I) for complete coding sequences, P1, P2, P3 region, and the 3D gene.The VP1 gene tree employed a gamma distribution (G).All trees underwent 1,000 replications of bootstrap analysis.Vietnamese CaKoV isolates are marked with red circle, and Thai CaKoV isolates are noted by blue square.

Figure 3 .
Figure 3.The physicochemical properties of canine kobuvirus (CaKoV) VP1 protein were evaluated using various methods: (a) Kolaskar & Tongaonkar antigenicity, (b) BepiPred linear epitope 2.0, (c) Chou & Fasman Beta-turn prediction, (d) Emini surface accessibility, (e) Karplus & Schulz flexibility prediction, and (f) Parker hydrophilicity.The Y-axis represents the corresponding score for each residue, while the X-axis corresponds to the residue positions in the sequence.

Figure 4 .
Figure 4.The presentation of B-cell epitopes derived from the VP1 capsid of canine kobuvirus (CaKoV) employing ElliPro server: (a-f) Predicted discontinuous B-cell epitopes, and (g) Predicted B-cell linear structure.

Table 1 .
Prevalence of canine kobuvirus (CaKoV) detection in Vietnam and Thailand by age of investigated dogs.-, no data available.

Table 2 .
Structure and length of the complete coding sequences (CDS) of canine kobuvirus (CaKoV) in Vietnam and Thailand.

Table 4 .
The selection sites of Vietnamese and Thai canine kobuvirus (CaKoV) based on VP1 region.†MEME: positive selection for Vietnamese strains at sites 204th and 206th; for Thai strains at sites 69th, 82nd, 138th and 206th.‡FUBAR: positive selection for Vietnamese strains at site 206th.Thailand (107 from healthy dogs and 107 from GID dogs).The RS samples were randomly collected from both healthy dogs and those with diarrhea.However, dogs with clinical manifestations of gastrointestinal issues due to food poisoning, hepatobiliary disease, or those vaccinated within the last 4 weeks were excluded from the study.Information about the sampled dogs, including age, breed, sex, and vaccination status, was recorded using a questionnaire.To analyze age-related risk factors associated with CaKoV detection, all dogs were categorized into six age groups, adjusted based on established age classifications for dogs